Protein structural information has proven beneficial for understanding structure/function relationships and for applications such as structure-based drug design. X-ray crystallography is the predominant technique used to obtain three-dimensional protein structure information. A critical component of this technique is the growth of high quality, well ordered crystals of the target protein. Advances in x-ray diffraction equipment, data collection methods, and computational capabilities have progressed to the point where the growth of high quality crystals is often the rate limiting step for the determination of three-dimensional protein structures. Many different techniques have been used in the attempt to grow high quality protein crystals. The most widely used protein crystal growth technique, vapor diffusion, utilizes a growth solution containing protein and a precipitating agent. A popular vapor diffusion configuration, typically described as the hanging-drop or Linbro method (McPherson, Jr., A. (1982), Preparation and Analysis of Protein Crystals (Wiley, New York)xe2x80x94see FIG. 1), uses a reservoir solution containing precipitant and a buffered protein/precipitant solution that xe2x80x9changsxe2x80x9d from a sealed cover slip positioned over the reservoir. The initial solution conditions are such that water vapor diffuses from the protein solution into the reservoir solution, thereby increasing the concentration of the protein beyond its solubility point. One significant limitation of the traditional vapor diffusion technique is that the evaporation of water from the growth solution (within a particular geometry) is fixed by the starting concentrations of the solution components (see FIG. 2). Thus, the rate at which the approach to supersaturation of the growth solution occurs is fixed, even if modification of this evaporation rate is desirable, and this technique suffers from the inability to control the vapor equilibration process once the experiment is initiated.
The vapor diffusion technique has been used successfully to grow protein crystals in the microgravity environment of NASA""s Space Shuttle, with space flight hardware called the Vapor Diffusion Apparatus or VDA, (Herrmainn, F. T., and Herren, B. J. (1990) xe2x80x9cCrystal Growth Apparatusxe2x80x9d, U.S. Pat. No. 4,919,899 and Snyder, R. S.; Herren, B. J.; Carter, D. C.; Yost, V. H.; Bugg, C. E.; DeLucas, L. J.; Suddath, F. L. (1991) xe2x80x9cMacromolecular Growing Sytemsxe2x80x9d, U.S. Pat. No. 5,013,531). The original Vapor Diffusion Apparatus (VDA) was used to grow protein crystals in the microgravity environment of NASA""s Space Shuttle. However, its concept and design have proven not to be optimal and it has specific limitations. For example, during crystal growth and nucleation, the vapor diffusion profile is fixed by the starting solution concentrations. Also no modification of the experiment is possible and photography during crystal growth is sporadic and non-isothermal. Although significant, the results with the Vapor Diffusion Apparatus are not optimal as evidenced by the following statistics. Only 25% of all proteins flown in the VDA produced crystals that diffracted better than any crystals grown on Earth. 40% of protein flown produced crystals which did not diffract better and 35% produced no crystals. Clearly there is need for methods and devices to improve the success ratio. Also, investigations have been underway (Smith, H. W. and DeLucas, L. J. (1991), J. Crystal Growth 110 137; Wilson, L. J. and Suddath, F. L. (1992), J. Crystal Growth 116 414) in the attempt to produce systems that will allow control over the evaporation profile of a growth solution. Early experiments showed that simply slowing down the evaporation rate of a growth solution generally produces a smaller population of larger crystals than can be obtained with traditional vapor diffusion techniques. Recent results from a large number of experiments shows this effect to be consistent not only for lysozyme, but for other proteins as well.
While vapor diffusion has been (and still is) a very popular technique, it has not always proven to be the best method for a given protein, and hence a wide range of other approaches have been used as a means to obtain high quality protein crystals. Another protein crystal growth technique, temperature, has only begun to be extensively explored in recent years. This technique utilizes the variable solubility versus temperature that some proteins exhibit for a given solution condition as a means for initiating and controlling crystal growth. Though this approach offers promise when compared to other techniques for controlling the rate of growth to produce high quality protein crystals, this method is not without limitations. Several proteins have been crystallized successfully using temperature (Baker, E. N. and Dodson, G. (1970), J. Mol. Biol. 54, 605; Shotton, D. M. , Hartley, B. S., Camerman, N. and Hofnab, T. (1968), J. Mol. Biol. 32, 155; Hanson, A. W., Applebury, M. L., Coleman, J. E. and Wyckoff, H. (1970), J. Biol. Chem. 245, 4975; McPherson, JR., A. and Rich, A. (1972), Biochem. Biophys. Acta 285, 493), and recent developments in custom instrumentation and devices that screen protein solubility versus temperature improve the usefulness of temperature as a strategic method for growing protein crystals (Cacioppo, E., Munson, S. and Pusey, M. L. (1991), J. Crystal Growth 110, 66).
Despite this, the approach to finding suitable conditions that yield high quality protein crystals predominantly has been a trial and error process, where more than one thousand crystallization conditions are typically screened, often without success. Several systems have been constructed to aid the growth of protein crystals. These systems vary in complexity from simple hand-held devices (Eisele, J.-L. (1993), J. Appl. Cryst. 26, 92) to complex robotic systems that simply prepare and monitor different conditions (Cox, M. J. and Weber, P. C. (1988), J. Crystal Growth 90, 318; Chayen, N. E., Stewart, P. D. S., Maeder, D. L. and Blow, D. M. (1990), J. Appl. Cryst. 23, 297). Only a few systems have attempted to achieve control over the dynamics of protein crystal growth (Wilson, L. J., Bray, T. L. and Suddath, F. L. (1991), J. Crystal Growth 110, 142; Casey, G. A. and Wilson, W. W. (1992), J. Crystal Growth 122, 95) by altering the rate at which water is removed from the growth solution. Other investigations have been underway (Smith, H. W. and DeLucas, L. J. (1991), J. Crystal Growth 110, 137; Wilson, L. J. and Suddath, F. L. (1992), J. Crystal Growth 116, 414) in the attempt to produce systems that will allow control over the evaporation profile of a growth solution. Early experiments showed that simply slowing down the evaporation rate of a growth solution generally produces a smaller population of larger crystals than can be obtained with traditional vapor diffusion techniques. However, they have not offered true dynamic control of the protein crystal growth process.
The present invention provides a system for dynamic control of crystal growth, particularly for difficult-to-crystallize macromolecular substances such as proteins, polypeptides, nucleic acids, viruses and virus fragments. The nucleic acids include DNA, RNA and fragments of DNA and RNA. While reference is made hereafter to protein crystal growth, it should be understood that these teachnigs will be equally applicable to other macromolecular substances. Dynamic control of protein crystal growth (DC/PCG) has operational advantages that include the ability to separate protein crystal aggregation and/or nucleation from the post nucleation protein crystal growth phase and the potential for limiting the number of nucleation sites. Supersaturation conditions necessary for the aggregation and /or nucleation of proteins are often significantly greater than those needed for subsequent growth of the crystal. In order to minimize problems during the subsequent growth phase caused by the higher supersaturation necessary for nucleation, it is desirable to separately control the nucleation and growth environments. With DC/PCG, one has the capability to vary post-nucleation growth kinetics. One also has the capability to optimize crystal growth conditions in subsequent experiments. With respect to microgravity experiments, DC/PCG also minimizes protein sample quantities and minimizes astronaut crew time in experiment operation.
The present invention provides systems with increased capacity and versatility for the growth of protein crystals by vapor diffusion or temperature. Temperature adjustments are advantageous in providing a non-invasive means of controlling the supersaturation environment of protein nuclei. Additionally, laser light scattering data from the growth medium allows the aggregation state of the protein to be evaluated non-invasively and provides for dynamic control of the crystallization process. These systems achieve truly dynamically controlled protein crystal growth system that can be used for both terrestrial and microgravity experiments.